The present invention relates to an inexpensive phytase with a low Michaelis constant (hereinafter abbreviated to Km) for phytic acid, which degrades phytic acid as an anti-trophic factor contained in feed, thereby improving the nutritive value of the feed and enabling an efficient utilization of phosphoric acid released by the degradation; and to a gene coding for the phytase.
Phosphorus is an essential element for all organisms. Plant-derived feeds used for the production of domestic animals contain phosphorus, 50 to 70% of which is present as phytic acid. Phytic acid is a major storage substance of phosphoric acid, existing in a large amount in plant seeds. However, phytic acid is excreted without digestion and absorption in the digestive organs in single-stomach animals such as pigs, chickens, etc. That is, phytic acid is a storage substance of phosphoric acid, but its phosphorus is not utilized at all. Accordingly, phosphoric acid is added to feed for single-stomach animals for the purpose of growth promotion.
Addition of phosphoric acid to the feed leads to an increase in the amount of phosphorus in excrement. In recent years, excrement from domestic animals increase considerably as the production of domestic animals increases more and more, whereby an environmental problem is now caused in the world. In particular, phosphorus contained in excrement is mentioned as a factor causing the phenomenon of nutrition enrichment in lakes and marshes, so the amount of phosphorus in excrement comes to be regulated, and there arises necessity for countermeasure.
In addition to the problem of excreted phosphorus, phytic acid chelates divalent metals important as a nutritive source, such as magnesium, calcium, zinc and iron, thereby making its absorption into animals difficult and reducing the nutritive value of feed. Accordingly, phytic acid is regarded as an anti-trophic factor.
From the foregoing, it has been examined to decrease the amount of phosphorus in excrement by treating the feed with a phytase known widely as an enzyme capable of hydrolyzing a salt of phytic acid into inositol and inorganic phosphoric acid in order to utilize phosphoric acid released from phytic acid in place of phosphoric acid conventionally added in feed, and it has also been examined to improve the nutritive value of the feed by decomposing phytic acid as an anti-trophic factor [U.S. Pat. No. 3,297,548 (1967); J. Nutrition, 101, 1289-1294 (1971)].
Known as phytase-producing microorganisms are bacteria such as Bacillus subtilis and Pseudomonas, yeasts such as Saccharomyces cerevisiae, and filamentous fungi such as Aspergillus terreus, Aspergillus ficcum and Aspergillus awamori. 
For phytase derived from Aspergillus ficcum, its purification and biochemical properties are described in Preparative Biochem., 18, 443-458 (1988), and its gene and amino acid sequence are described in Gene, 127, 87-94 (1993).
For phytase derived from Aspergillus awamori, its nucleotide sequence and amino acid sequence are described in Gene, 133, 55-62 (1993).
Michaelis constants (Km) for phytases known so far are 0.57 mM for wheat bran-derived phytase [Agr. Biol. Chem., 26, 794-803 (1962)], 0.17 mM for rice bran-derivedphytase [Agr. Biol. Chem., 53, 1475-1483 (1898)], 117 xcexcM for maize (Zea mays)-derived phytase, 250 xcexcM for Aspergillus ficcum-derived phytase (WO 91/05053), 330 xcexcM for Aspergillus oryzae-derived phytase, 150 xcexcM for Bacillus subtilis-derived phytase, 500 xcexcM for Bacillus natto-derived phytase, and 130 xcexcM for Escherichia coli-derived phytase.
To demonstrate the performance of the enzyme, the concentration of a substrate is necessary to be higher than Km, and if an enzyme with low Km and an enzyme with high Km have the same maximum reaction rate (Vmax), the enzyme with low Km does not decrease a reaction rate even at a lower substrate concentration as compared with the enzyme with high Km.
That is, when compared with the enzyme with high Km, the enzyme with low Km is advantageous in that a sufficient degradation rate can be achieved even at a lower substrate concentration, thereby minimizing the amount of the remaining substrate.
Accordingly, there is a demand for an inexpensive phytase with a low Km value for phytic acid, which phytase degrades phytic acid being an anti-trophic factor contained in feed, thereby improving the nutritive value of the feed and simultaneously achieving an efficient utilization of phosphoric acid released by the degradation.
The present invention relates to a phytase (hereinafter referred to as xe2x80x9cnovel phytasexe2x80x9d) having a Michaelis constant of 10 to 30 xcexcM when using phytic acid as the substrate, DNA coding for the phytase, recombinant DNA having the DNA introduced thereinto, a transformant carrying the recombinant DNA, a process for preparing a phytase by use of the transformant, and an animal feed containing the phytase.
The present invention will be described in detail.
A specific example of the novel phytase of the present invention includes: a phytase with the following physicochemical properties:
(1) Km value: 10 to 30 xcexcM;
(2) molecular weight (by SDS-PAGE): about 60 kDa after treatment with endoglycosidase H;
(3) optimum pH: pH 5.0 to 6.5;
(4) optimum temperature: 45 to 65xc2x0 C. showing maximum activity;
(5) substrate specificity: acting on the substrates, phytic acid, p-nitrophenylphosphate, D-glucose 6-phosphate, fructose 6-phosphate, D-myo-inositol 1,4,5-triphosphate, glycerol phosphate, and adenosine triphosphate; and
(6) isoelectric focusing: pI 4.7 to 5.4.; or
a phytase protein having the amino acid sequence shown in SEQ ID NO:1 or 2.
Furthermore, the present invention encompasses a novel phytase having an amino acid sequence (for example, shown in SEQ ID NO:3) in which a secretory signal sequence has been linked to the novel phytase described above.
The present invention further includes a phytase having an amino acid sequence comprising substitutions, deletions or additions of one or more amino acids relative to the amino acid sequence shown in SEQ ID No:1, 2 or 3; having a homology of 40% or more to the amino acid sequence shown in SEQ ID NO:1, 2 or 3; and having a Michaelis constant (Km) of 10 to 30 xcexcM when using phytic acid as the substrate. The phytase has preferably a homology of 60% or more, more preferably 80% or more.
The substitution, deletion or addition of amino acids can be carried out according to methods described in Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci., USA, 79, 6409 (1982), Proc. Natl. Acad. Sci., USA, 81, 5662 (1984), Science, 224, 1431 (1984), PCT WO85/00817 (1985), Nature, 316, 601 (1985), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Current Protocols in Molecular Biology, chapter eight xe2x80x9cMutagenesis of Cloned DNAxe2x80x9d, John Wiley and Sons, Inc. (1989), etc.
The novel phytase can also be obtained from any microorganisms having the ability to produce it. Among them, preferable examples are microorganisms belonging to the genus Aspergillus and having the ability to produce the novel phytase. More preferable examples include Aspergillus niger strain SK57 (FERM BP-5473) or mutants or derived strains thereof. Aspergillus niger strain SK92 (FERM BP-5481) is included in mutants derived from Aspergillus niger strain SK57.
The gene (hereinafter referred to as xe2x80x9cnovel phytase genexe2x80x9d) coding for the novel phytase of the present invention may be any gene coding for the novel phytase: for example, a gene coding for a phytase having the amino acid sequence shown in SEQ ID NO:1, 2 or 3; or a gene coding for a phytase which has an amino acid sequence where in the amino acid sequence shown in SEQ ID NO:1, 2 or 3, one or more amino acids have been substituted, deleted or added and which has a Michaelis constant (Km) of 10 to 30 xcexcM when using phytic acid as the substrate. The gene may contain introns in the DNA sequence. Specifically, the gene of the present invention includes DNA shown in SEQ ID NO:4, or DNA shown in SEQ ID NO:5 containing introns in its sequence.
Further, the novel phytase gene of the present invention includes DNA capable of hybridizing under stringent conditions with the above-defined DNA, and of bringing about the corresponding phytase activity.
The term xe2x80x9cDNA capable of hybridizing under stringent conditionsxe2x80x9d as described herein refers to DNA obtainable using colony hybridization, plaque hybridization or Southern blot hybridization wherein any DNA contained in the base sequence shown in SEQ ID NO:4 or 5 is used as a probe. A specific example thereof is DNA which can be identified by subjecting it to hybridization with a colony or plaque derived DNA-immobilized filter in the presence 0.7 to 1.0 M NaCl at 65xc2x0 C. and then washing the filter at 65xc2x0 C. with a 0.1 to 2xc3x97SSC solution (1xc3x97SSC solution is composed of 150 mM sodium chloride and 15 mM sodium citrate).
Hybridization can be effected according to methods described in Molecular Cloning, A Laboratory Manual, 2nd ed., Sambrook, Fritsch, Maniatis, Cold Spring Harbor Laboratory Press (1989) (hereinafter abbreviated to xe2x80x9cMolecular Cloning, 2nd ed.xe2x80x9d). Specifically, the DNA capable of hybridizing includes DNA having a homology of 60% or more, preferably 80% or more, more preferably 95% or more to the base sequence of SEQ ID NO:4 or 5.
A DNA fragment containing the novel phytase gene can be obtained from any microorganisms having the ability to produce novel phytase. Although any microorganism having the ability to produce novel phytase can be used, preferable examples are microorganisms belonging to the genus Aspergillus and having the ability to produce novel phytase, more preferably Aspergillus niger strain SK57 or mutants or derived strains thereof. Aspergillus niger strain SK92 is included in mutants of Aspergillus niger strain SK57.
Aspergillus niger strain SK57 was deposited as FERM BP-5473 on Mar. 22, 1996 and Aspergillus niger strain SK92 as FERM BP-5481 on Mar. 12, 1996, respectively, with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, 305 Japan.
Further, an animal feed comprising the novel phytase is included in the present invention.
Hereinafter, the method of obtaining the phytase gene derived from microorganisms having the ability to produce the novel phytase will be described.
Chromosomal DNA is prepared from microorganisms having the ability to produce the novel phytase by using a conventional DNA isolation method, for example, the phenol method [Biochim. Biophys. Acta., 72, 619 (1963)]. The resulting chromosomal DNA is cleaved with suitable restriction enzymes, and these fragments cleaved with the restriction enzymes are introduced into vector DNA to construct a genomic DNA library from the microorganism. This DNA library is used to transform a host microorganism. The resulting transformants are selected for a transfornant containing the novel phytase gene through hybridization. A DNA containing the gene of interest can be obtained from the selected transformant.
A series of these procedures can follow in vitro recombination techniques known in the art (Molecular Cloning, 2nd ed.).
The vector DNA for constructing the genomic cDNA library of the microorganism having the ability to produce novel phytase may be any of phage vectors, plasmid vectors, etc. which are autonomously replicable in E. coli strain K12. Examples thereof are ZAP Express [Strategies, 5, 58 (1992); Stratagene], pBluescript II SK(+) [Nucleic Acids Research, 17, 9494 (1989), Stratagene], xcexzap II (Stratagene), xcexgt10, xcexgt11 [DNA Cloning, A Practical Approach, 1, 49 (1985)], Lambda BlueMid (Clonetech), xcexExCell (Pharmacia), pT7 T3 18U (Pharmacia), pcD2 [Mol. Cell. Biol., 3, 280 (1983)] and pUC18 [Gene, 33, 103 (1985)].
The host microorganism may be any microorganism belonging to the genus Escherichia. Examples thereof are Escherichia coli XL1-Blue MRFxe2x80x2 [Strategies, 5, 81 (1992); Stratagene], Escherichia coli C600 [Genetics, 39, 440 (1954)], Escherichia coli Y1088 [Science, 222, 778 (1983)], Escherichi coli Y1090 [Science, 222, 778 (1983)], Eschericia coli NM522 [J. Mol. Biol., 166, 1 (1983)], Escherichia coli K802 [J. Mol. Biol., 16, 118 (1996)], Escherichia coli JM105 [Gene, 38, 275 (1985)], Escherichia coli JM109, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, etc.
The transformant carrying the novel phytase gene can be selected by hybridization.
The probe useful in hybridization includes an oligonucleotide synthesized on the basis of a partial amino acid sequence determined for novel phytase. If another phytase gene has already been obtained from a class of microorganism closely related to the microorganism having the ability to produce novel phytase, the -gene can be used in some case as the probe for the novel phytase gene. The gene which can be used in such case as the probe includes a phytase gene from Aspergillus ficcum. 
The phytase gene from Aspergillus ficcum can be obtained by preparing its chromosomal DNA according to the above-described method and amplifying its phytase gene by polymerase chain reaction (PCR) with DNA primers which have been synthesized based on the DNA sequence of the phytase gene from Aspergillus ficcum. 
The DNA primers can be synthesized using a conventional DNA synthesizer such as a DNA synthesizer (Shimadzu Seisakusho, Japan) utilizing the thiophosphite method, a DNA synthesizer model 392 (Perkin Elmer) or 380A DNA synthesizer (Applied Systems) utilizing the phosphoamidite method. Examples of the DNA primer synthesized in this manner include DNAs shown in SEQ ID NOS:6 and 7.
The DNA containing the novel phytase gene obtained from the transformant selected by hybridization is analyzed by the method such as the dideoxy method of Sanger et al. [Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)], whereby the base sequence of the gene can be determined. The analysis of the base sequence can also be effected using an automatic base sequence analyzer such as SQ-5500 DNA sequencer (Hitachi) or 373A DNA sequencer [Perkin Elmer].
The thus determined nucleotide sequence of the novel phytase gene includes e.g. the nucleotide sequence of SEQ ID NO:4 or 5.
For expression in hosts, the novel phytase gene thus obtained may be expressed by the method described in Molecular Cloning, 2nd ed. or Current Protocols in Molecular Biology Supplements 1-34.
First, the DNA fragment containing the novel phytase gene is cleaved with restriction enzymes or DNase to form DNA fragments having a suitable size containing the novel phytase gene, then inserted into a region downstream of a promoter in an expression vector, followed by introducing the expression vector into which the DNA has been inserted, into a host compatible with the expression vector.
Any host can be used insofar as it can express the gene of interest. Examples thereof include prokaryotes belonging to the genera Escherichia, Serratia, Corynebacterium, Brevibacterium, Pseudomonas, Bacillus, Microbacterium, etc.; filamentous fungi belonging to the genera Aspergillus, Rhizopus, Trichoderma, Neurospora, Mucor, Penicillium, etc.; yeasts belonging to the genera Kluyvermyces, Saccharomyces, Schizosaccharomyces, Trichosporon, Schwanniomyces, etc.; animal cells; and insect cells.
The expression vector used is one capable of autonomously replicating in the above host or capable of integrating into the chromosome, containing a promoter at a site enabling transcription of the novel phytase gene.
If prokaryotes such as bacteria are used as the host, the expression vector for novel phytase is preferably one capable of autonomously replicating in the microorganism and comprising a promoter, a ribosome-binding sequence, the novel phytase gene, and a transcription termination sequence. The vector may also contain a gene for regulating the promoter.
Expression vectors include e.g. pKK233-2 (Pharmacia), pSE280 (Invitrogen), pGEMEX-1 (Promega), pQE-8 (Qiagen), pKYP10 (JP-A-110600/1983), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci., USA, 82, 4306 (1985)], pBluescript (Stratagene), pTrs30 [prepared from Eschericha coli JM109/pTrS30 (FERM BP-5407)], pTrs32 [prepared from Escherichia coli JM109/pTrS32 (FERM BP-5408)], pGHA2 [E. coli containing pGHA2 has been deposited as Escherichia coli IGHA2 (FERM BP-400) with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Japan; see JP-A-221091/1985], pGKA2 [E. coli containing pGKA2 has been deposited as Escherichia coli IGKA2 (FERM B-6798) with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, JP-A-221091/1985], pTerm2 (JP-A-22979/1991, U.S. Pat. No. 4,686,191, U.S. Pat. No. 4,939,094, U.S. Pat. No. 5,160,735), PGEX (Pharmacia), pET system (Novagen), and pSupex.
Any promoter can be used insofar as it can be expressed in a host such as E. coli. Examples thereof are promoters derived from E. coli, phage etc., such as trp promoter (Ptrp), lac promoter (Plac), PL promoter and PR promoter. Further, artificially modified promoters are usable, whose examples are a Ptrpxc3x972 promoter (having 2 Ptrp promoters in series), tac promoter, T7 promoter, PletI promoter, etc.
The ribosome-binding sequence used is preferably a plasmid in which the distance between a Shine-Dalgarno sequence and an initiation codon is suitably regulated (e.g. distance of 6 to 18 bases).
Although any gene coding for the novel phytase can be used as the novel phytase gene, its nucleotides are preferably replaced such that the DNA sequence of the gene is composed of optimum codons for expression in host microorganisms.
Although a transcription termination sequence is not necessarily required for expression of the gene of the present invention, it is preferable to locate the transcription termination sequence at a site just downstream of the structural gene.
The host includes microorganisms belonging to the genera Escherichia, Serratia, Corynebacterium, Brevibacterium, Pseudomonas, Bacillus, etc., for example, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli hB101, Escherichia coli No. 49, Escherichia coli W3110, Escherichia coli NY49, Bacillus subtilis, Bacillus amyloliquefacines, Brevibacterium immariophilum ATCC14068, Brevibacterium saccharolyticum ATCC14066, Brevibacterium flavum ATCC14067, Brevibacterium lactofermentum ATCC13869, Corynebacterium glutamicum ATCC13032, Corynebacterium acetoacidophilum ATCC13870, and Microbacterium ammoniaphilum ATCC15354.
If filamentous fungi are used as the host, examples of expression vectors are p3SR2 [Gene, 26, 205-221 (1983)], pKBY2 [Proc. Natl. Acad. Sci. USA, 82, 834-838 (1985)], pSal23 [Agric. Biol. Chem., 51, 2549-2555 (1987)], pSTA14 [Mol. Gen. Genet., 218, 99-104 (1989)], pDJB2 [Gene, 36, 321-331 (1989)], and pLeu4 [Biosci. Biotech. Biochem., 56, 1503-1504 (1992)].
Any promoter can be used insofar as it allows expression to induce in filamentous fungi as the host. Examples are a promoter induced strongly by starch or cellulose, e.g. a promoter for glucoamylase or xcex1-amylase from the genus Aspergillus or cellulase (cellobiohydrase) from the genus Trichoderma, a promoter for enzymes in the glycolytic pathway, such as phosphoglycerate kinase (pgk) and glycerylaldehyde 3-phosphate dehydrogenase (gpd), etc.
Although any gene coding for the novel phytase can be used as the novel phytase gene, a preferable example is a gene coding for a protein having an amino acid sequence to which a peptide having a secretory signal sequence at an N-terminal amino acid of the novel phytase has been linked to permit secretion of the novel phytase out of the microorganism cell. The peptide having a secretory signal sequence includes e.g. a peptide having a secretory signal sequence for glucoamylase or xcex1-amylase from the genus Aspergillua, or a peptide having the 1-24 amino acid sequence shown in SEQ ID NO:2.
The host includes Aspergillus niger SK57, Aspergillus oryzae M-2-3 [Agric. Biol. Chem., 51, 2549-2555 (1987)]; Aspergillius ficcum NRRL3135 , Aspergillus awamori NRRL3112, Aspergillius nidulans IFO4340, Trichoderma reesei Rut-C-30 [Appl. Microbiol. Biotechnol., 20, 46-53 (1984)], Rhizopus niveus M-37 [Biosci. Biotech. Biochem., 56, 1503-1504 (1992)], etc.
Transformation of filamentous fungi can be performed according to the method of Gomi et al. [Agric. Biol. Chem., 51, 2549 (1987)] or the like.
If yeasts are used as the host, expression vectors such as YEp 13 (ATCC37115), YEp 24 (ATCC37051) and YCp 50 (ATCC37419) can be enumerated.
Any promoter capable of expressing in yeast hosts can be used as the promoter. Examples thereof include promoters for genes of hexokinase and the like in the glycolytic pathway, and promoters such as gal 1 promoter, gal 10 promoter, heat shock protein promoter, MFxcex1 1 promoter and CUP 1 promoter.
Examples of the host are Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans, Schwanniomyces alluvius, etc.
Introduction of the recombinant vector can be carried out by any method of introducing DNA into yeasts, such as electroporation method [Methods, Enzymol., 194, 182 (1990)], spheroplast method [Proc. Natl. Aad. Sci. USA, 84, 1929 (1978)], lithium acetate method [J. Bacteriol., 153, 163 (1983)] and the like.
If animal cells are used as the host, expression vectors used are, e.g., pAGE107 [JP-A-22979/1991; Cytotechnology, 3, 133 (1990)], pAS3-3 (JP-A-227075/1990), pCDM8 [Nature, 329, 840 (1987)], pcDNAI/Amp (Invitrogen) , pREP4 (Invitrogen) , pAGE103 [J. Biochem., 101, 1307 (1987)], and pAGE210.
Any promoter which allows expression to induce in animal cells can be used. Examples thereof are a promoter for an IE (immediate early) gene in cytomegalovirus (human CMV), an SV40 early promoter, and a promoter for metallothionein. Also, an enhancer for the IE gene from human CMV may be used together with the promoter.
The host cells include Namalwa cell that is a human cell, COS cell that is a monkey cell, CHO cell that is a Chinese hamster cell, HBT5637 (JP-A-299/1988), etc.
Any method capable of introducing DNA into animal cells can be used: for example, electroporation method [Cytotechnology, 3, 133 (1990)], calcium phosphate method (JP-A-227075/1990), lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)], etc.
Preparation and culture of the transformant can be conducted according to the methods described in JP-A-227075/1990 or JP-A-257891/1990.
If an insect cell is used as the host, a gene for the protein of interest can be expressed by the methods described in e.g. Baculovirus Expression Vectors, A Laboratory Manual; Current Protocols in Molecular Biology Supplements 1-34; and Bio/Technology, 6, 47 (1988).
That is, the vector into which the recombinant gene has been introduced is introduced together with baculovirus into an insect cell so that a recombinant virus is obtained in the supernatant of the cultured insect cell. Then, insect cells are infected with the recombinant virus whereby the protein can be expressed.
The gene-introducing vector used in this method includes e.g. pLV1392, pVL1393, and pBlueBacIII (which all are products of Invitrogen)
As the baculovirus, it is possible to employ e.g. Autographa californica nuclear polyhedrosis virus, which is a virus infecting certain moth insects.
As the insect cells, it is possible to employ ovary cells Sf9 and Sf21 from Spodoptera frugiperda [Baculovirus Expression Vectors, A Laboratory Manual, W. H. Freeman and Company, New York (1992)], High 5 (Invitrogen) which is an ovary cell from Trichoplusia ni, etc.
For co-introduction of both the vector having the recombinant gene and the baculovirus into an insect cell to prepare a recombinant virus, the calcium phosphate method (JP-A-227075/1990) or lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)] may be used.
Expression of the gene may be performed in a secretion manner or as expression of a fusion protein in accordance with the methods described in Molecular Cloning, 2nd ed., in addition to direct expression.
In the case of expression in filamentous fungi, yeasts, animal cells or insect cells, polypeptides having saccharide or carbohydrate chains can be obtained.
Besides the above-described transformants, a microorganism having the ability to produce the novel phytase or its mutants having more improved ability to produce the novel phytase can be used to produce the novel phytase.
The mutant having the improved productivity of the novel phytase can be obtained through usual mutagenesis.
For preparation of the novel phytase, the microorganism having the ability to produce the novel phytase, mutants derived from the microorganism, or transformants carrying the recombinant DNA having the novel phytase gene integrated therein can be cultured by a conventional culture method to produce and accumulate the novel phytase, followed by recovering of the novel phytase from the culture. The microorganisms, mutants, and transformants used for producing the novel phytase are hereinafter referred to as novel phytase-producing organism.
If the novel phytase-producing organism is a prokaryote such as E. coli or a eukaryote such as filamentous fungous or yeast, the medium for culturing these organisms may be natural or synthetic insofar as the medium contains a carbon source, a nitrogen source, inorganic salts, and so on, which can be assimilated by the organisms and in which the transformants can be efficiently cultured.
Any carbon source can be used insofar as it can be assimilated by the microorganisms: for example, hydrocarbons such as glucose, fructose, sucrose, molasses containing them, starch, and starch hydrolysates; organic acids such as acetic acid and propionic acid; and alcohols such as methanol, ethanol and propanol.
Used as the nitrogen source are: ammonia; ammonium salts of inorganic or organic acids, such as ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate; and other nitrogenous compounds; peptone; meat extract (broth); yeast extract; corn steep liquor; casein hydrolysates; soybean cake and hydrolysates thereof; and a variety of fermentation microorganisms and digested materials thereof.
The inorganic matter used include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, etc.
For culture of filamentous fungi, a medium with wheat bran, rice bran etc. as carbon, nitrogen and inorganic sources supplemented with suitable salts can also be used.
Culture is conducted under aerobic conditions using shake culture or submerged shake culture under aeration. The culture temperature is preferably 15 to 40xc2x0 C., and the culture time is usually 16 to 96 hours. During culture, pH is kept at 3.0 to 9.0, and in culture of filamentous fungi, pH is kept preferably at 3.0 to 6.5. pH is adjusted with an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like.
During culture, an antibiotic such as ampicillin or tetracycline may optionally be added to the medium.
If a microorganism transformed with an expression vector having an inducible promoter as the promoter is cultured, its inducer may optionally be added to the medium. For example, isopropyl-xcex2-D-thiogalactopyranoside (IPTG) or the like may be added to the medium for culture of a microorganism transformed with an expression vector having a lac promoter, and indoleacrylic acid (IAA) or the like may be added to the medium for culture of a microorganism transformed with an expression vector having a trp promoter.
If a filamentous fungus is cultured in a medium containing a solid component such as wheat bran, the filamentous fungus is inoculated and then well mixed with the solid component until they become uniform, after which the mixture is spread thinly on a number of aluminum or stainless steel trays, put in a cellar, and cultured under the control of temperature, humidity and aeration. More specifically, the fungus is subjected to stationary culture at 25 to 35xc2x0 C. for 3 to 10 days under 100% humidity in a culture chamber.
If the novel phytase-producing organism is an animal cell, the medium used for culturing the cell is a generally used medium such as RPMI1640 medium, Eagle""s MEM medium, or a medium supplemented with fetal calf serum or the like to said medium.
Culture may be conducted in the presence of 5% CO2. The culture temperature is preferably 35 to 37xc2x0 C., and the culture time is usually 3 to 7 days.
During culture, an antibiotic such as kanamycin or penicillin may optionally be added to the medium.
The medium for culture of the transformant prepared from an insect host cell may be a normal medium such as TNM-FH medium (Pharmingen) , Sf-900 II SFM medium (Gibco BRL), and ExCell 400 and ExCell 405 [both, JRH Biosciences].
The culture temperature is preferably 25 to 30xc2x0 C., and the culture time is usually 1 to 4 days.
During culture, an antibiotic such as gentamicin may optionally be added to the medium.
For isolation and purification of the novel phytase from a culture of the novel phytase-producing organism, the conventional isolation/purification of enzymes can be used.
For example, if the novel phytase is accumulated in a soluble form in cells of the novel phytase-producing organism, the cells are collected from the culture by centrifugation, then washed with an aqueous buffer and disrupted by ultrasonication using a French press, manntongaurin homogenizer, dynomill or the like, thereby giving a cell-free extract. The supernatant is obtained by centrifuging the cell-free extract and then subjected to the conventional isolation/purification of enzymes: namely, solvent extraction, salting-out with amonium sulfate, desalting precipitation with organic solvent, anion-exchange chromatography on resin such as diethylaminoethyl (DEAE)-Sepharose or DIAION HPA-75 (Mitsubishi Corporation), anion-exchange chromatography on resin such as S-Sepharose FF (Pharmacia), hydrophobic chromatography on resin such as butyl Sepharose or phenyl Sepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, and electrophoresis such as isoelectric focusing, which means can be used singly or in combination, whereby a purified preparation can be obtained.
If the novel phytase is expressed in an insoluble form within cells, then, the cells are similarly recovered, disrupted and centrifuged to give a precipitated fraction from which the novel phytase is then recovered, and the insoluble novel phytase is solubilized with a detergent for polypeptide. The resultant liquid is then diluted or dialyzed to the degree that the detergent is not contained or does not cause denaturation of the polypeptide, whereby the novel phytase is reconstituted in the normal conformation. And a purified preparation can be obtained by the isolation/purification as described above.
If the novel phytase is secreted out of cells, the culture is centrifuged to give a soluble fraction. If ingredients in the medium contain a solid component such as wheat bran, the novel phytase can be extracted with warm water or the like and subjected to centrifugation to give a soluble fraction. From the soluble fraction, a purified preparation of the novel phytase can be obtained in the same way for isolation and purification from the cell-free extract as described above.
The activity of the novel phytase can be determined according to the standard assay method described below.
0.5 ml of 0.2 M acetate buffer, pH 5.5 (sodium acetate) containing 2.5 mM sodium phytate (Sigma) is maintained at 37xc2x0 C. for 5 minutes, and 0.5 ml of an enzyme solution is added to initiate the reaction. After maintained at 37xc2x0 C. for 10 minutes, 2 ml of a stop solution of enzyme reaction (i.e., mixture of 1:1:2 10 mM ammonium molybdate, 5 N sulfuric acid and acetone) is added to stop the reaction, and 0.1 ml of 1 M citric acid is further added and mixed. The absorbance of this solution is determined at 380 nm on a spectrophotometer (Hitachi U-2000). One unit of phytase activity is defined as the amount of enzyme allowing to release 1 xcexcmol inorganic phosphorus for 1 minute at pH 5.5 at 37xc2x0 C.
The Km value of the novel phytase can be determined by the Lineweaver-Burk plot in which the activity of the novel phytase, as determined by the standard assay, is plotted at varying concentrations of the substrate.
The novel phytase of the present invention can be utilized in various processes required for converting a salt of phytic acid into inositol and inorganic phosphoric acid.
For example, the present enzyme can be used in animal feeds, soybean processing, liquid feeds for pigs and poultry, and production of inositol or inositol monophosphate from salts of phytic acid.
An example of such animal feeds is as follows:
The novel phytase of the present invention is mixed with a carrier material such as wheat chaff and dried in a spray tower or a fluidized bed. After drying, an osmotic pressure stabilizer such as sorbitol, and a preservative, such as benzoic acid, are further added to give an animal feed. The amount of the novel phytase in the animal feed is 10 to 5000 U, preferably 100 to 1000 U per kg of the animal feed.